Nucleic acids encoding human NIK protein

ABSTRACT

The invention provides methods and compositions relating to a novel kinase, NIK, involved in NFκB activation. The polypeptides may be produced recombinantly from transformed host cells from the disclosed NIK encoding nucleic acids or purified from human cells. The invention provides isolated NIK hybridization probes and primers capable of specifically hybridizing with the disclosed NIK genes, NIK-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

FIELD OF THE INVENTION

The field of this invention is proteins involved in transcription factoractivation.

BACKGROUND

Cytokines trigger changes in gene expression by modifying the activityof otherwise latent transcription factors (Hill and Treisman, 1995).Nuclear factor κB (NF-κB) is a prominent example of how such an externalstimulus is converted into an active transcription factor (Verma et al.,1995). The NF-κB system is composed of homo- and heterodimers of membersof the Rel family of related transcription factors that control theexpression of numerous immune and inflammatory response genes as well asimportant viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel,1994). The activity of NF-κB transcription factors is regulated by theirsubcellular localization (Verma et al., 1995). In most cell types, NF-κBis present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit.This heterodimer is sequestered in the cytoplasm in association withIκBα a member of the IκB family of inhibitory proteins (Finco andBaldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). IκBαmasks the nuclear localization signal of NF-κB and thereby preventsNF-κB nuclear translocation. Conversion of NF-κB into an activetranscription factor that translocates into the nucleus and binds tocognate DNA sequences requires the phosphorylation and subsequentubiquitin-dependent degradation of IκBα in the 26s proteasome.Signal-induced phosphorylation of IκBα occurs at serines 32 and 36.Mutation of one or both of these serines renders IκBα resistant toubiquitination and proteolytic degradation (Chen et al., 1995);DiDonato, 1996 #370, Roff, 1996 #397.

The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1(IL-1) are among the physiological inducers of IκB phosphorylation andsubsequent NF-κB activation (Osborn et al., 1989; Beg et al., 1993).Although TNF and IL-1 initiate signaling cascades leading to NF-κBactivation via distinct families of cell-surface receptors (Smith etal., 1994; Dinarello, 1996), both pathways utilize members of the TNFreceptor-associated factor (TRAF) family of adaptor proteins as signaltransducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b).TRAF proteins were originally found to associate directly with thecytoplasmic domains of several members of the TNF receptor familyincluding the 75 kDa TNF receptor (TNFR2), CD40, CD30, and thelymphotoxin-β receptor (Rothe et al., 1994; Hu et al., 1994; Cheng etal., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al.,1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). Inaddition, TRAF proteins are recruited indirectly to the 55 kDa TNFreceptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996).Activation of NF-κB by TNF requires TRAF2 (Rothe et al., 1995; Hsu etal., 1996). TRAF5 has also been implicated in NF-κB activation bymembers of the TNF receptor family (Nakano et al., 1996); Ishida, 1996#240. In contrast, TRAF6 participates in NF-κB activation by IL-1 (Caoet al., 1996b). Upon IL-1 treatment, TRAF6 associates with IRAK, aserine-threonine kinase that binds to the IL-1 receptor complex (Cao etal., 1996a); Huang, 1997 #400.

An NF-κB-inducing kinase (NIK), a member of the MAP kinase kinase kinase(MAP3K) family, was identified as a TRAF2-interacting protein (Malininet al., 1997). NIK activates NF-κB when overexpressed, andkinase-inactive mutants of NIK comprising its TRAF2-interactingC-terminal domain (NIK.sub.(624-947)) or lacking two crucial lysineresidues in its kinase domain (NIK.sub.(KK429-430AA)) behave asdominant-negative inhibitors that suppress TNF-, IL-1-, andTRAF2-induced NF-κB activation (Malinin et al., 1997).

Here, we disclose a novel human NIK(NIK .sub.(Ala25), which alsoprovides the foregoing functionalities yet deviates in terms of criticalsequence and structural characteristics; in particular, a Pro-Alasubstitution at position 25 imposes altered protein structure. We showthat the NIK.sub.(Ala25) variant interacts with and cross-phosphorylatesthe IκB Kinases α and β, IKK-α and IKK-β (see Goeddel et al. and Rotheet al., copending applications T97-006 and T97-007, respectively, filedJul. 1, 1997). IKK-α and IKK-β have sequence similarity to theconceptual translate of a previously identified open reading framepostulated to encode a serine-threonine kinase of unknown function(`Conserved Helix-loop-helix Ubiquitous Kinase` or CHUK, Connelly andMarcu, 1995; Mock et al., 1995). Catalytically inactive mutants of theIKKs suppress NF-κB activation induced by TNF and IL-1 stimulation aswell as by TRAF and NIK overexpression; transiently expressed IKKsassociate with the endogenous IκBα complex; and the IKKs phosphorylateIκBα on serines 32 and 36. As used herein, Ser32 and Ser36 of IκB referscollectively to the two serine residues which are part of the consensussequence DSGL/IXSM/L (e.g. ser 32 and 36 in IκBα, ser 19 and 23 in IκBβ,and ser 157 and 161, or 18 and 22, depending on the usage ofmethionines, in IκBε, respectively. In addition, we disclose thatNIK.sub.(Ala25) associates with other members of the TRAF family,including TRAF5 and TRAF6. Catalytically inactive mutants ofNIK.sub.(Ala25) also inhibit TRAF5- and TRAF6-induced NF-κB activation,thus providing a unifying concept for NIK.sub.(Ala25) as a commonmediator in the NF-κB signaling cascades triggered by TNF and IL-1downstream of TRAFs.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to isolated NIKpolypeptides, related nucleic acids, polypeptide domains thereof havingNIK-specific structure and activity and modulators of NIK function,particularly IKKκ/α kinase activity. NIK polypeptides can regulate NFκBactivation and hence provide important regulators of cell function. Thepolypeptides may be produced recombinantly from transformed host cellsfrom the subject NIK polypeptide encoding nucleic acids or purified frommammalian cells. The invention provides isolated NIK hybridizationprobes and primers capable of specifically hybridizing with thedisclosed NIK gene, NIK-specific binding agents such as specificantibodies, and methods of making and using the subject compositions indiagnosis (e.g. genetic hybridization screens for NIK transcripts),therapy (e.g. NIK kinase inhibitors to inhibit TNF signal transduction)and in the biopharmaceutical industry (e.g. as immunogens, reagents forisolating other transcriptional regulators, reagents for screeningchemical libraries for lead pharmacological agents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of a natural human cDNA encoding a human NIKpolypeptide is shown as SEQ ID NO: 1, and the fall conceptual translateis shown as SEQ ID NO:2. This novel NIK cDNA sequence was cloned by PCRusing primers designed from GenBank accesion number Y102565. The NIKpolypeptides of the invention include incomplete translates of SEQ IDNO: 1 which translates and deletion mutants of SEQ ID NO:2 have humanNIK-specific amino acid sequence, binding specificity or function andcomprise Ala25. Preferred translates/deletion mutants comprise at leasta 10 residue Ala25-containing domain of SEQ ID NO:2, preferablyincluding residues 22-31, more preferably including residues 12-31, mostpreferably including residues 2-31. The subject domains provide NIKdomain specific activity or function, such as NIK-specific kinase orkinase inhibitory activity, IKK-α/β (SEQ ID NO:3/4,respectively)-binding or binding inhibitory activity, TRAF1, 2, 3, 5and/or 6 binding or binding inhibitory activity, IκB-binding or bindinginhibitory activity, NFκB activating or inhibitory activity or antibodybinding. Preferred domains phosphorylate at least one serine residue ofIKK-α and/or β.

NIK-specific activity or finction may be determined by convenient invitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cellculture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.Binding assays encompass any assay where the molecular interaction of anNIK polypeptide with a binding target is evaluated. The binding targetmay be a natural intracellular binding target such as an NIK substrate,a NIK regulating protein or other regulator that directly modulates NIKactivity or its localization; or non-natural binding target such aspecific immune protein such as an antibody, or an NIK specific agentsuch as those identified in screening assays such as described below.NIK-binding specificity may assayed by kinase activity or bindingequilibrium constants (usually at least about 10⁷ M⁻¹, preferably atleast about 10⁸ M⁻¹, more preferably at least about 10⁹ M⁻¹), by theability of the subject polypeptide to function as negative mutants inNIK-expressing cells, to elicit NIK specific antibody in a heterologoushost (e.g a rodent or rabbit), etc. In any event, the NIK bindingspecificity of the subject NIK polypeptides necessarily distinguishesthat of the human NIK protein of Malinin et al. (1997).

The claimed NIK polypeptides are isolated or pure: an "isolated"polypeptide is unaccompanied by at least some of the material with whichit is associated in its natural state, preferably constituting at leastabout 0.5%, and more preferably at least about 5% by weight of the totalpolypeptide in a given sample and a pure polypeptide constitutes atleast about 90%, and preferably at least about 99% by weight of thetotal polypeptide in a given sample. The NIK polypeptides andpolypeptide domains may be synthesized, produced by recombinanttechnology, or purified from mammalian, preferably human cells. A widevariety of molecular and biochemical methods are available forbiochemical synthesis, molecular expression and purification of thesubject compositions, see e.g. Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et aL, Greene Publ. Assoc.,Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides binding agents specific to the claimed NIKpolypeptides, including substrates, agonists, antagonists, naturalintracellular binding targets, etc., methods of identifying and makingsuch agents, and their use in diagnosis, therapy and pharmaceuticaldevelopment. For example, specific binding agents are useful in avariety of diagnostic and therapeutic applications, especially wheredisease or disease prognosis is associated with improper utilization ofa pathway involving the subject proteins, e.g. NF-κB activation. NovelNIK-specific binding agents include NIK-specific receptors, such assomatically recombined polypeptide receptors like specific antibodies orT-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory) and other naturalintracellular binding agents identified with assays such as one-, two-and three-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc. Agents of particular interest modulate NIK function, e.g.NIK-dependent transcriptional activation. For example, a wide variety ofinhibitors of NIK IKK-α/β kinase activity may be used to regulate signaltransduction involving IκB. Exemplary NIK kinase inhibitors includeknown classes of serine/threonine kinase (e.g. PKC) inhibitors such ascompetitive inhibitors of ATP and substrate binding, antibiotics,NIK-derived peptide inhibitors, esp. dominant negative deletion mutants,etc., see Tables 1 and 2. NIK specificity and activity are readilyquantified in high throughput kinase assays using panels of proteinkinases (see cited references and Examples).

Preferred inhibitors include natural compounds such as staurosporine(Omura S, et al. J Antibiot (Tokyo) 1995 July;48(7):535-48), produced bya marine organism, and synthetic compounds such as PD 153035, which alsopotently inhibits the EGF receptor protein kinase (Fry DW et al. Science1994 Aug. 19;265(5175):1093-5). Members of the tyrphostin family ofsynthetic protein kinase inhibitors are also useful; these includecompounds which are pure ATP competitors, compounds which are puresubstrate competitors, and compounds which are mixed competitors:compete with both ATP and substrate (Levitzki A and Gazit A, Science1995 Mar. 24;267(5205):1782-8). Additional NIK inhibitors includepeptide-based substrate competitors endogenously made by the mammaliancell, e.g. PKI (protein kinase inhibitor, Seasholtz A.F. et al., ProcNatl Acad Sci USA 1995 Feb. 28;92(5):173-8 ), or proteins inhibiting cdckinases (Correa-Bordes J and Nurse P, Cell 1995 Dec. 15;83(6):1001-9).Additional small peptide based substrate competitive kinase inhibitorsand allosteric inhibitors (inhibitory mechanisms independent of ATP orsubstrate competition) are readily generated by established methods(Hvalby O et al. Proc Natl Acad Sci USA 1994 May 24;91(11):4761-5; BaijaP, et al., Cell Immunol 1994 January153(1):28-38; Villar-Palasi C,Biochim Biophys Acta 1994 Dec. 30;1224(3):384-8; Liu W. Z., et al.,Biochemistry 1994 Aug. 23;33(33):10120-6).

                  TABLE I                                                         ______________________________________                                        Selected Small Molecule NIK Kinase Inhibitors                                 Inhibitors  Citations                                                         ______________________________________                                        HA-100.sup.1                                                                              1. Hagiwara, M,. et al. Mol. Pharmacol. 32:7                                  (1987)                                                            Chelerythrine.sup.2                                                                       2. Herbert, J. M., et al. Biochem Biophys Res                                 Com 172:993 (1990)                                                Staurosporin.sup.3,4,5                                                                    3. Schachtele, C., et al. Biochem Biophys Res                                 Com 151:542 (1988)                                                Calphostin C.sup.6,7,8,9                                                                  4. Tamaoki, T., et al. Biochem Biophys Res                                    Com 135:397 (1986)                                                K-252b.sup.10                                                                             5. Tischler, A. S., et al. J. Neurochemistry                                  55:1159 (1990)                                                    PCK 19-36.sup.11                                                                          6. Bruns, R. F., et al. Biochem Biophys Res                                   Com 176:288 (1991)                                                Iso-H7.sup.12                                                                             7. Kobayashi, E., et al. Biochem Biophys Res                                  Com 159:548 (1989)                                                PKC 19-31   8. Tamaoki, T., et al Adv 2nd Mass                                            Phosphoprotein Res 24:497 (1990)                                  H-7.sup.13,3,14                                                                           9. Tamaoki, T., et al. Biotechnology 8:732                                    (1990)                                                            H-89.sup.15 10. Yasuzawa, T. J. Antibiotics 39:172 (1986)                     KT5720.sup.16                                                                             11. House, C., et al. Science 238:1726 (1987)                     cAMP-depPKinhib.sup.17                                                                    12. Quick, J., et al. Biochem. Biophys. Res.                                  Com. 167:657 (1992)                                               A-3.sup.18  13. Bouli, N. M. and Davis, M. Brain Res.                                     525:198 (1990)                                                    HA1004.sup.19,20                                                                          14. Takahashi, I., et al. J. Pharmacol. Exp.                                  Ther. 255:1218 (1990)                                             K-252a.sup.16,5                                                                           15. Chijiwa, T., et al. J. Biol. Chem.                                        265:5267 (1990)                                                   KT5823.sup.16                                                                             16. Kase, H., et al. Biochem. Biophys. Res.                                   Com. 142:436 (1987)                                               ML-9.sup.21 17. Cheng, H. C., et al. J. Biol. Chem. 261:989                               (1986)                                                            KT5926.sup.22                                                                             18. Inagaki, M., et al. Mol. Pharmacol. 29:577                                (1986)                                                                        19. Asano, T. and Hidaka, H. J Pharmaco. Exp                                  Ther 231:141 (1984)                                                           20. Hidaka, H., et al. Biochemistry 23:5036                                   (1984)                                                                        21. Nagatsu, T., et al. Biochem Biophys Res                                   Com 143:1045 (1987)                                                           22. Nakanishi, S., et al. Mol. Pharmacol. 37:482                              (1990)                                                            ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Selected Peptidyl NIK Kinase Inhibitors                                       ______________________________________                                        hIKKα, residues 2-398                                                                    NIK, residues 624-947                                        hIKKα, residues 279-547                                                                  NIK, residues 1-645, Ala429, Ala430                          hIκBβ, residues 5-381                                                               TRAF2, residues 225-501                                      hIκBβ, residues 301-641                                                             TRAF6, residues 218-512                                      ______________________________________                                    

Accordingly, the invention provides methods for modulating signaltransduction involving IκB in a cell comprising the step of modulatingNIK kinase activity, e.g. by contacting the cell with a serine/threoninekinase inhibitor. The cell may reside in culture or in situ, i.e. withinthe natural host. Preferred inhibitors are orally active in mammalianhosts. For diagnostic uses, the inhibitors or other NIK binding agentsare frequently labeled, such as with fluorescent, radioactive,chemiluminescent, or other easily detectable molecules, eitherconjugated directly to the binding agent or conjugated to a probespecific for the binding agent.

The amino acid sequences of the disclosed NIK polypeptides are used toback-translate NIK polypeptide-encoding nucleic acids optimized forselected expression systems Holler et al. (1993) Gene 136, 323-328;Martin et al. (1995) Gene 154, 150-166) or used to generate degenerateoligonucleotide primers and probes for use in the isolation of naturalNIK-encoding nucleic acid sequences ("GCG" software, Genetics ComputerGroup, Inc, Madison Wis.). NIK-encoding nucleic acids used inNIK-expression vectors and incorporated into recombinant host cells,e.g. for expression and screening, transgenic animals, e.g. forfunctional studies such as the efficacy of candidate drugs for diseaseassociated with NIK-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a NIK cDNA specific sequencecomprising SEQ ID NO: 1, bases 72-75, and sufficient to effect specifichybridization thereto (i.e. specifically hybridize with SEQ ID NO: 1 inthe presence of the NIK cDNA described by Malinin et al. (1997). Suchprimers or probes are at least 12, preferably at least 24, morepreferably at least 36 and most preferably at least 96 bases in length.Demonstrating specific hybridization generally requires stringentconditions, for example, hybridizing in a buffer comprising 30%formamide in 5×SSPE (0.18M NaCl, 0.01M NaPO₄, pH7.7, 0.001M EDTA) bufferat a temperature of 42° C. and remaining bound when subject to washingat 42° C. with 0.2×SSPE; preferably hybridizing in a buffer comprising50% formamide in 5×SSPE buffer at a temperature of 42° C. and remainingbound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C.NIK nucleic acids can also be distinguished using alignment algorithms,such as BLASTX (Altschul et al. (1990) Basic Local Alignment SearchTool, J Mol Biol 215, 403-410).

The subject nucleic acids are of synthetic/non-natural sequences and/orare isolated, i.e. unaccompanied by at least some of the material withwhich it is associated in its natural state, preferably constituting atleast about 0.5%, preferably at least about 5% by weight of totalnucleic acid present in a given fraction, and usually recombinant,meaning they comprise a non-natural sequence or a natural sequencejoined to nucleotide(s) other than that which it is joined to on anatural chromosome. Recombinant nucleic acids comprising the nucleotidesequence of SEQ ID NO:1, or fragments thereof comprising SEQ ID NO:1,bases 72-75, contain such sequence or fragment at a terminus,immediately flanked by (i.e. contiguous with) a sequence other than thatwhich it is joined to on a natural chromosome, or flanked by a nativeflanking region fewer than 10 kb, preferably fewer than 2 kb, which isat a terminus or is immediately flanked by a sequence other than thatwhich it is joined to on a natural chromosome. While the nucleic acidsare usually RNA or DNA, it is often advantageous to use nucleic acidscomprising other bases or nucleotide analogs to provide modifiedstability, etc.

The subject nucleic acids find a wide variety of applications includinguse as translatable transcripts, hybridization probes, PCR primers,diagnostic nucleic acids, etc.; use in detecting the presence of NIKgenes and gene transcripts and in detecting or amplifying nucleic acidsencoding additional NIK homologs and structural analogs. In diagnosis,NIK hybridization probes find use in identifying wild-type and mutantNIK alleles in clinical and laboratory samples. Mutant alleles are usedto generate allele-specific oligonucleotide (ASO) probes forhigh-throughput clinical diagnoses. In therapy, therapeutic NIK nucleicacids are used to modulate cellular expression or intracellularconcentration or availability of active NIK.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of a NIKmodulatable cellular function. Generally, these screening methodsinvolve assaying for compounds which modulate NIK interaction with anatural NIK binding target such as IKKα and/or β, TRAF1, 2, 3, 5 or 6,etc. A wide variety of assays for binding agents are provided includinglabeled in vitro protein-protein binding assays, immunoassays, cellbased assays, etc. The methods are amenable to automated, cost-effectivehigh throughput screening of chemical libraries for lead compounds.Identified reagents find use in the pharmaceutical industries for animaland human trials; for example, the reagents may be derivatized andrescreened in in vitro and in vivo assays to optimize activity andminimize toxicity for pharmaceutical development.

In vitro binding assays employ a mixture of components including an NIKpolypeptide, which may be part of a fusion product with another peptideor polypeptide, e.g. a tag for detection or anchoring, etc. The assaymixtures comprise a natural intracellular NIK binding target. In aparticular embodiment, the binding target is a a IKKα and/or β-derivedsubstrate of NIK kinase activity. Such substrates comprise aNIK-phosphoylatable IKKα and/or β serine residue and at least 5,preferably at least 10, and more preferably at least 20 naturallyoccurring immediately flanking residues on each side. While nativefull-length binding targets may be used, it is frequently preferred touse portions (e.g. peptides) thereof so long as the portion providesbinding affinity and avidity to the subject NIK polypeptide convenientlymeasurable in the assay. The assay mixture also comprises a candidatepharmacological agent. Candidate agents encompass numerous chemicalclasses, though typically they are organic compounds; preferably smallorganic compounds and are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. A variety ofother reagents may also be included in the mixture. These includereagents like ATP or ATP analogs (for kinase assays), salts, buffers,neutral proteins, e.g. albumin, detergents, protease inhibitors,nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharmacological agent, the NIK polypeptidespecifically binds the cellular binding target, portion or analog with areference binding affinity. The mixture components can be added in anyorder that provides for the requisite bindings and incubations may beperformed at any temperature which facilitates optimal binding.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the NIK polypeptideand one or more binding targets is detected by any convenient way. ForNIK kinase assays, `binding` is generally detected by a change in thephosphorylation of a NIK substrate. In this embodiment, kinase activitymay quantified by the transfer to the substrate of a labeled phosphate,where the label may provide for direct detection as radioactivity,luminescence, optical or electron density, etc. or indirect detectionsuch as an epitope tag, etc. A variety of methods may be used to detectthe label depending on the nature of the label and other assaycomponents, e.g. through optical or electron density, radiativeemissions, nonradiative energy transfers, etc. or indirectly detectedwith antibody conjugates, etc.

A difference in the binding affinity of the NIK polypeptide to thetarget in the absence of the agent as compared with the binding affinityin the presence of the agent indicates that the agent modulates thebinding of the NIK polypeptide to the NIK binding target. Analogously,in the cell-based assay also described below, a difference inNIK-dependent transcriptional activation in the presence and absence ofan agent indicates the agent modulates NIK function. A difference, asused herein, is statistically significant and preferably represents atleast a 50%, more preferably at least a 90% difference.

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The following experimental section and examples are offered by way ofillustration and not by way of limitation.

EXAMPLES

1. Protocol for at NIK-IKK-β phosphorylation assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

kinase: 10⁻⁸ -10⁻⁵ M NIK kinase domain deletion mutant (SEQ ID NO:2,residues 2-644) at 20 μg/ml in PBS.

substrate: 10⁻⁷ -10⁻⁴ M biotinylated IKK-β (SEQ ID NO:4) substrate at 40μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.4,0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktailof protease inhibitors.

³² P!γ-ATP 10×stock: 2×10⁻⁵ M cold ATP with 100 μCi ³² P!γ-ATP. Place inthe 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 40 μl biotinylated substrate (2-200 pmoles/40 ul in assay buffer)

Add 40 μl kinase (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Add 10 μl ³² P!γ-ATP 10×stock.

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. cold ATP at 80% inhibition.

2. Protocol for high throughput NIKI-TRAF2 binding assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 MM MgCl₂, 1% glycerol,0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of proteaseinhibitors.

³³ P NIK polypeptide 10×stock: 10⁻⁸ -10⁻⁶ M "cold" NIK supplemented with200,000-250,000 cpm of labeled NIK (Beckinan counter). Place in the 4°C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (B3MB #1017128), 10 mg APMSF (BMB #917575),and 2 mM NaVO₃ (Sigma #S-6508) in 10 ml of PBS.

TRAF2: 10⁻⁷ ×10⁵ M biotinylated TRAF2 in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-NIK (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹ -10⁻⁷ M finalconc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated TRAF2 (0.1-10 pmoles/40 μl in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated TRAF2) at 80% inhibition.

3. Protocol for high throughput IμB-complex formation assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol,0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of proteaseinhibitors.

³³ P NIK polypeptide 10×stock: 10⁻⁸ -10⁻⁶ M "cold" NIK supplemented with200,000-250,000 cpm of labeled NIK (Beckman counter). Place in the 4° C.microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVO₃ (Sigma #S-6508) in 10 ml of PBS.

IκB: 10⁷ -10⁵ M biotinylated IκB in PBS.

IKK-β: 10⁻⁷ 10⁵ M in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-NIK (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹ -10⁻⁷ M finalconc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 20 μM IKK-β(0.1-10 pmoles/20 ul in assay buffer)

Add 20 μM biotinylated IκB (0.1-10 pmoles/20 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated IκB) at 80% inhibition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3156 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGCAGTGATGGAAATGGCCTGCCCAGGTGCCCCTGGCTCAGCAGTGGGGCAGCAGAAG60                GAACTCCCCAAAGCCAAGGAGAAGACGCCGCCACTGGGGAAGAAACAGAGCTCCGTCTAC120               AAGCTTGAGGCCGTGGAGAAGAGCCCTGTGTTCTGCGGAAAGTGGGAGATCCTGAATGAC180               GTGATTACCAAGGGCACAGCCAAGGAAGGCTCCGAGGCAGGGCCAGCTGCCATCTCTATC240               ATCGCCCAGGCTGAGTGTGAGAATAGCCAAGAGTTCAGCCCCACCTTTTCAGAACGCATT300               TTCATCGCTGGGTCCAAACAGTACAGCCAGTCCGAGAGTCTTGATCAGATCCCCAACAAT360               GTGGCCCATGCTACAGAGGGCAAAATGGCCCGTGTGTGTTGGAAGGGAAAGCGTCGCAGC420               AAAGCCCGGAAGAAACGGAAGAAGAAGAGCTCAAAGTCCCTGGCTCATGCAGGAGTGGCC480               TTGGCCAAACCCCTCCCCAGGACCCCTGAGCAGGAGAGCTGCACCATCCCAGTGCAGGAG540               GATGAGTCTCCACTCGGCGCCCCATATGTTAGAAACACCCCGCAGTTCACCAAGCCTCTG600               AAGGAACCAGGCCTTGGGCAACTCTGTTTTAAGCAGCTTGGCGAGGGCCTACGGCCGGCT660               CTGCCTCGATCAGAACTCCACAAACTGATCAGCCCCTTGCAATGTCTGAACCACGTGTGG720               AAACTGCACCACCCCCAGGACGGAGGCCCCCTGCCCCTGCCCACGCACCCCTTCCCCTAT780               AGCAGACTGCCTCATCCCTTCCCATTCCACCCTCTCCAGCCCTGGAAACCTCACCCTCTG840               GAGTCCTTCCTGGGCAAACTGGCCTGTGTAGACAGCCAGAAACCCTTGCCTGACCCACAC900               CTGAGCAAACTGGCCTGTGTAGACAGTCCAAAGCCCCTGCCTGGCCCACACCTGGAGCCC960               AGCTGCCTGTCTCGTGGTGCCCATGAGAAGTTTTCTGTGGAGGAATACCTAGTGCATGCT1020              CTGCAAGGCAGCGTGAGCTCAAGCCAGGCCCACAGCCTGACCAGCCTGGCCAAGACCTGG1080              GCAGCACGGGGCTCCAGATCCCGGGAGCCCAGCCCCAAAACTGAGGACAACGAGGGTGTC1140              CTGCTCACTGAGAAACTCAAGCCAGTGGATTATGAGTACCGAGAAGAAGTCCACTGGGCC1200              ACGCACCAGCTCCGCCTGGGCAGAGGCTCCTTCGGAGAGGTGCACAGGATGGAGGACAAG1260              CAGACTGGCTTCCAGTGCGCTGTCAAAAAGGTGCGGCTGGAAGTATTTCGGGCAGAGGAG1320              CTGATGGCATGTGCAGGATTGACCTCACCCAGAATTGTCCCTTTGTATGGAGCTGTGAGA1380              GAAGGGCCTTGGGTCAACATCTTCATGGAGCTGCTGGAAGGTGGCTCCCTGGGCCAGCTG1440              GTCAAGGAGCAGGGCTGTCTCCCAGAGGACCGGGCCCTGTACTACCTGGGCCAGGCCCTG1500              GAGGGTCTGGAATACCTCCACTCACGAAGGATTCTGCATGGGGACGTCAAAGCTGACAAC1560              GTGCTCCTGTCCAGCGATGGGAGCCACGCAGCCCTCTGTGACTTTGGCCATGCTGTGTGT1620              CTTCAACCTGATGGCCTGGGAAAGTCCTTGCTCACAGGGGACTACATCCCTGGCACAGAG1680              ACCCACATGGCTCCGGAGGTGGTGCTGGGCAGGAGCTGCGACGCCAAGGTGGATGTCTGG1740              AGCAGCTGCTGTATGATGCTGCACATGCTCAACGGCTGCCACCCCTGGACTCAGTTCTTC1800              CGAGGGCCGCTCTGCCTCAAGATTGCCAGCGAGCCTCCGCCTGTGAGGGAGATCCCACCC1860              TCCTGCGCCCCTCTCACAGCCCAGGCCATCCAAGAGGGGCTGAGGAAAGAGCCCATCCAC1920              CGCGTGTCTGCAGCGGAGCTGGGAGGGAAGGTGAACCGGGCACTACAGCAAGTGGGAGGT1980              CTGAAGAGCCCTTGGAGGGGAGAATATAAAGAACCAAGACATCCACCGCCAAATCAAGCC2040              AATTACCACCAGACCCTCCATGCCCAGCCGAGAGAGCTTTCGCCAAGGGCCCCAGGGCCC2100              CGGCCAGCTGAGGAGACAACAGGCAGAGCCCCTAAGCTCCAGCCTCCTCTCCCACCAGAG2160              CCCCCAGAGCCAAACAAGTCTCCTCCCTTGACTTTGAGCAAGGAGGAGTCTGGGATGTGG2220              GAACCCTTACCTCTGTCCTCCCTGGAGCCAGCCCCTGCCAGAAACCCCAGCTCACCAGAG2280              CGGAAAGCAACCGTCCCGGAGCAGGAACTGCAGCAGCTGGAAATAGAATTATTCCTCAAC2340              AGCCTGTCCCAGCCATTTTCTCTGGAGGAGCAGGAGCAAATTCTCTCGTGCCTCAGCATC2400              GACAGCCTCTCCCTGTCGGATGACAGTGAGAAGAACCCATCAAAGGCCTCTCAAAGCTCG2460              CGGGACACCCTGAGCTCAGGCGTACACTCCTGGAGCAGCCAGGCCGAGGCTCGAAGCTCC2520              AGCTGGAACATGGTGCTGGCCCGGGGGCGGCCCACCGACACCCCAAGCTATTTCAATGGT2580              GTGAAAGTCCAAATACAGTCTCTTAATGGTGAACACCTGCACATCCGGGAGTTCCACCGG2640              GTCAAAGTGGGAGACATCGCCACTGGCATCAGCAGCCAGATCCCAGCTGCAGCCTTCAGC2700              TTGGTCACCAAAGACGGGCAGCCTGTTCGCTACGACATGGAGGTGCCAGACTCGGGCATC2760              GACCTGCAGTGCACACTGGCCCCTGATGGCAGCTTCGCCTGGAGCTGGAGGGTCAAGCAT2820              GGCCAGCTGGAGAACAGGCCCTAACCCTGCCCTCCACCGCCGGCTCCACACTGCCGGAAA2880              GCAGCCTTCCTGCTCGGTGCACGATGCTGCCCTGAAAACACAGGCTCAGCCGTTCCCAGG2940              GGATTGCCAGCCCCCCGGCTCACAGTGGGAACCAGGGCCTCGCAGCAGCAAGGTGGGGGC3000              AAGCAGAATGCCTCCCAGGATTTCACACCTGAGCCCTGCCCCACCCTGCTGAAAAAACAT3060              CCGCCACGTGAAGAGACAGAAGGAGGATGGCAGGAGTTACCTGGGGAAACAAAACAGGGA3120              TCTTTTTCTGCCCCTGCTCCAGTCGAGTTGGCCTGA3156                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 947 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaValMetGluMetAlaCysProGlyAlaProGlySerAlaVal                              151015                                                                        GlyGlnGlnLysGluLeuProLysAlaLysGluLysThrProProLeu                              202530                                                                        GlyLysLysGlnSerSerValTyrLysLeuGluAlaValGluLysSer                              354045                                                                        ProValPheCysGlyLysTrpGluIleLeuAsnAspValIleThrLys                              505560                                                                        GlyThrAlaLysGluGlySerGluAlaGlyProAlaAlaIleSerIle                              65707580                                                                      IleAlaGlnAlaGluCysGluAsnSerGlnGluPheSerProThrPhe                              859095                                                                        SerGluArgIlePheIleAlaGlySerLysGlnTyrSerGlnSerGlu                              100105110                                                                     SerLeuAspGlnIleProAsnAsnValAlaHisAlaThrGluGlyLys                              115120125                                                                     MetAlaArgValCysTrpLysGlyLysArgArgSerLysAlaArgLys                              130135140                                                                     LysArgLysLysLysSerSerLysSerLeuAlaHisAlaGlyValAla                              145150155160                                                                  LeuAlaLysProLeuProArgThrProGluGlnGluSerCysThrIle                              165170175                                                                     ProValGlnGluAspGluSerProLeuGlyAlaProTyrValArgAsn                              180185190                                                                     ThrProGlnPheThrLysProLeuLysGluProGlyLeuGlyGlnLeu                              195200205                                                                     CysPheLysGlnLeuGlyGluGlyLeuArgProAlaLeuProArgSer                              210215220                                                                     GluLeuHisLysLeuIleSerProLeuGlnCysLeuAsnHisValTrp                              225230235240                                                                  LysLeuHisHisProGlnAspGlyGlyProLeuProLeuProThrHis                              245250255                                                                     ProPheProTyrSerArgLeuProHisProPheProPheHisProLeu                              260265270                                                                     GlnProTrpLysProHisProLeuGluSerPheLeuGlyLysLeuAla                              275280285                                                                     CysValAspSerGlnLysProLeuProAspProHisLeuSerLysLeu                              290295300                                                                     AlaCysValAspSerProLysProLeuProGlyProHisLeuGluPro                              305310315320                                                                  SerCysLeuSerArgGlyAlaHisGluLysPheSerValGluGluTyr                              325330335                                                                     LeuValHisAlaLeuGlnGlySerValSerSerSerGlnAlaHisSer                              340345350                                                                     LeuThrSerLeuAlaLysThrTrpAlaAlaArgGlySerArgSerArg                              355360365                                                                     GluProSerProLysThrGluAspAsnGluGlyValLeuLeuThrGlu                              370375380                                                                     LysLeuLysProValAspTyrGluTyrArgGluGluValHisTrpAla                              385390395400                                                                  ThrHisGlnLeuArgLeuGlyArgGlySerPheGlyGluValHisArg                              405410415                                                                     MetGluAspLysGlnThrGlyPheGlnCysAlaValLysLysValArg                              420425430                                                                     LeuGluValPheArgAlaGluGluLeuMetAlaCysAlaGlyLeuThr                              435440445                                                                     SerProArgIleValProLeuTyrGlyAlaValArgGluGlyProTrp                              450455460                                                                     ValAsnIlePheMetGluLeuLeuGluGlyGlySerLeuGlyGlnLeu                              465470475480                                                                  ValLysGluGlnGlyCysLeuProGluAspArgAlaLeuTyrTyrLeu                              485490495                                                                     GlyGlnAlaLeuGluGlyLeuGluTyrLeuHisSerArgArgIleLeu                              500505510                                                                     HisGlyAspValLysAlaAspAsnValLeuLeuSerSerAspGlySer                              515520525                                                                     HisAlaAlaLeuCysAspPheGlyHisAlaValCysLeuGlnProAsp                              530535540                                                                     GlyLeuGlyLysSerLeuLeuThrGlyAspTyrIleProGlyThrGlu                              545550555560                                                                  ThrHisMetAlaProGluValValLeuGlyArgSerCysAspAlaLys                              565570575                                                                     ValAspValTrpSerSerCysCysMetMetLeuHisMetLeuAsnGly                              580585590                                                                     CysHisProTrpThrGlnPhePheArgGlyProLeuCysLeuLysIle                              595600605                                                                     AlaSerGluProProProValArgGluIleProProSerCysAlaPro                              610615620                                                                     LeuThrAlaGlnAlaIleGlnGluGlyLeuArgLysGluProIleHis                              625630635640                                                                  ArgValSerAlaAlaGluLeuGlyGlyLysValAsnArgAlaLeuGln                              645650655                                                                     GlnValGlyGlyLeuLysSerProTrpArgGlyGluTyrLysGluPro                              660665670                                                                     ArgHisProProProAsnGlnAlaAsnTyrHisGlnThrLeuHisAla                              675680685                                                                     GlnProArgGluLeuSerProArgAlaProGlyProArgProAlaGlu                              690695700                                                                     GluThrThrGlyArgAlaProLysLeuGlnProProLeuProProGlu                              705710715720                                                                  ProProGluProAsnLysSerProProLeuThrLeuSerLysGluGlu                              725730735                                                                     SerGlyMetTrpGluProLeuProLeuSerSerLeuGluProAlaPro                              740745750                                                                     AlaArgAsnProSerSerProGluArgLysAlaThrValProGluGln                              755760765                                                                     GluLeuGlnGlnLeuGluIleGluLeuPheLeuAsnSerLeuSerGln                              770775780                                                                     ProPheSerLeuGluGluGlnGluGlnIleLeuSerCysLeuSerIle                              785790795800                                                                  AspSerLeuSerLeuSerAspAspSerGluLysAsnProSerLysAla                              805810815                                                                     SerGlnSerSerArgAspThrLeuSerSerGlyValHisSerTrpSer                              820825830                                                                     SerGlnAlaGluAlaArgSerSerSerTrpAsnMetValLeuAlaArg                              835840845                                                                     GlyArgProThrAspThrProSerTyrPheAsnGlyValLysValGln                              850855860                                                                     IleGlnSerLeuAsnGlyGluHisLeuHisIleArgGluPheHisArg                              865870875880                                                                  ValLysValGlyAspIleAlaThrGlyIleSerSerGlnIleProAla                              885890895                                                                     AlaAlaPheSerLeuValThrLysAspGlyGlnProValArgTyrAsp                              900905910                                                                     MetGluValProAspSerGlyIleAspLeuGlnCysThrLeuAlaPro                              915920925                                                                     AspGlySerPheAlaTrpSerTrpArgValLysHisGlyGlnLeuGlu                              930935940                                                                     AsnArgPro                                                                     945                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 745 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetGluArgProProGlyLeuArgProGlyAlaGlyGlyProTrpGlu                              151015                                                                        MetArgGluArgLeuGlyThrGlyGlyPheGlyAsnValCysLeuTyr                              202530                                                                        GlnHisArgGluLeuAspLeuLysIleAlaIleLysSerCysArgLeu                              354045                                                                        GluLeuSerThrLysAsnArgGluArgTrpCysHisGluIleGlnIle                              505560                                                                        MetLysLysLeuAsnHisAlaAsnValValLysAlaCysAspValPro                              65707580                                                                      GluGluLeuAsnIleLeuIleHisAspValProLeuLeuAlaMetGlu                              859095                                                                        TyrCysSerGlyGlyAspLeuArgLysLeuLeuAsnLysProGluAsn                              100105110                                                                     CysCysGlyLeuLysGluSerGlnIleLeuSerLeuLeuSerAspIle                              115120125                                                                     GlySerGlyIleArgTyrLeuHisGluAsnLysIleIleHisArgAsp                              130135140                                                                     LeuLysProGluAsnIleValLeuGlnAspValGlyGlyLysIleIle                              145150155160                                                                  HisLysIleIleAspLeuGlyTyrAlaLysAspValAspGlnGlySer                              165170175                                                                     LeuCysThrSerPheValGlyThrLeuGlnTyrLeuAlaProGluLeu                              180185190                                                                     PheGluAsnLysProTyrThrAlaThrValAspTyrTrpSerPheGly                              195200205                                                                     ThrMetValPheGluCysIleAlaGlyTyrArgProPheLeuHisHis                              210215220                                                                     LeuGlnProPheThrTrpHisGluLysIleLysLysLysAspProLys                              225230235240                                                                  CysIlePheAlaCysGluGluMetSerGlyGluValArgPheSerSer                              245250255                                                                     HisLeuProGlnProAsnSerLeuCysSerLeuIleValGluProMet                              260265270                                                                     GluAsnTrpLeuGlnLeuMetLeuAsnTrpAspProGlnGlnArgGly                              275280285                                                                     GlyProValAspLeuThrLeuLysGlnProArgCysPheValLeuMet                              290295300                                                                     AspHisIleLeuAsnLeuLysIleValHisIleLeuAsnMetThrSer                              305310315320                                                                  AlaLysIleIleSerPheLeuLeuProProAspGluSerLeuHisSer                              325330335                                                                     LeuGlnSerArgIleGluArgGluThrGlyIleAsnThrGlySerGln                              340345350                                                                     GluLeuLeuSerGluThrGlyIleSerLeuAspProArgLysProAla                              355360365                                                                     SerGlnCysValLeuAspGlyValArgGlyCysAspSerTyrMetVal                              370375380                                                                     TyrLeuPheAspLysSerLysThrValTyrGluGlyProPheAlaSer                              385390395400                                                                  ArgSerLeuSerAspCysValAsnTyrIleValGlnAspSerLysIle                              405410415                                                                     GlnLeuProIleIleGlnLeuArgLysValTrpAlaGluAlaValHis                              420425430                                                                     TyrValSerGlyLeuLysGluAspTyrSerArgLeuPheGlnGlyGln                              435440445                                                                     ArgAlaAlaMetLeuSerLeuLeuArgTyrAsnAlaAsnLeuThrLys                              450455460                                                                     MetLysAsnThrLeuIleSerAlaSerGlnGlnLeuLysAlaLysLeu                              465470475480                                                                  GluPhePheHisLysSerIleGlnLeuAspLeuGluArgTyrSerGlu                              485490495                                                                     GlnMetThrTyrGlyIleSerSerGluLysMetLeuLysAlaTrpLys                              500505510                                                                     GluMetGluGluLysAlaIleHisTyrAlaGluValGlyValIleGly                              515520525                                                                     TyrLeuGluAspGlnIleMetSerLeuHisAlaGluIleMetGluLeu                              530535540                                                                     GlnLysSerProTyrGlyArgArgGlnGlyAspLeuMetGluSerLeu                              545550555560                                                                  GluGlnArgAlaIleAspLeuTyrLysGlnLeuLysHisArgProSer                              565570575                                                                     AspHisSerTyrSerAspSerThrGluMetValLysIleIleValHis                              580585590                                                                     ThrValGlnSerGlnAspArgValLeuLysGluArgPheGlyHisLeu                              595600605                                                                     SerLysLeuLeuGlyCysLysGlnLysIleIleAspLeuLeuProLys                              610615620                                                                     ValGluValAlaLeuSerAsnIleLysGluAlaAspAsnThrValMet                              625630635640                                                                  PheMetGlnGlyLysArgGlnLysGluIleTrpHisLeuLeuLysIle                              645650655                                                                     AlaCysThrGlnSerSerAlaArgSerLeuValGlySerSerLeuGlu                              660665670                                                                     GlyAlaValThrProGlnAlaTyrAlaTrpLeuAlaProAspLeuAla                              675680685                                                                     GluHisAspHisSerLeuSerCysValValThrProGlnAspGlyGlu                              690695700                                                                     ThrSerAlaGlnMetIleGluGluAsnLeuAsnCysLeuGlyHisLeu                              705710715720                                                                  SerThrIleIleHisGluAlaAsnGluGluGlnGlyAsnSerMetMet                              725730735                                                                     AsnLeuAspTrpSerTrpLeuThrGlu                                                   740745                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 756 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerTrpSerProSerLeuThrThrGlnThrCysGlyAlaTrpGlu                              151015                                                                        MetLysGluArgLeuGlyThrGlyGlyPheGlyAsnValIleArgTrp                              202530                                                                        HisAsnGlnGluThrGlyGluGlnIleAlaIleLysGlnCysArgGln                              354045                                                                        GluLeuSerProArgAsnArgGluArgTrpCysLeuGluIleGlnIle                              505560                                                                        MetArgArgLeuThrHisProAsnValValAlaAlaArgAspValPro                              65707580                                                                      GluGlyMetGlnAsnLeuAlaProAsnAspLeuProLeuLeuAlaMet                              859095                                                                        GluTyrCysGlnGlyGlyAspLeuArgLysTyrLeuAsnGlnPheGlu                              100105110                                                                     AsnCysCysGlyLeuArgGluGlyAlaIleLeuThrLeuLeuSerAsp                              115120125                                                                     IleAlaSerAlaLeuArgTyrLeuHisGluAsnArgIleIleHisArg                              130135140                                                                     AspLeuLysProGluAsnIleValLeuGlnGlnGlyGluGlnArgLeu                              145150155160                                                                  IleHisLysIleIleAspLeuGlyTyrAlaLysGluLeuAspGlnGly                              165170175                                                                     SerLeuCysThrSerPheValGlyThrLeuGlnTyrLeuAlaProGlu                              180185190                                                                     LeuLeuGluGlnGlnLysTyrThrValThrValAspTyrTrpSerPhe                              195200205                                                                     GlyThrLeuAlaPheGluCysIleThrGlyPheArgProPheLeuPro                              210215220                                                                     AsnTrpGlnProValGlnTrpHisSerLysValArgGlnLysSerGlu                              225230235240                                                                  ValAspIleValValSerGluAspLeuAsnGlyThrValLysPheSer                              245250255                                                                     SerSerLeuProTyrProAsnAsnLeuAsnSerValLeuAlaGluArg                              260265270                                                                     LeuGluLysTrpLeuGlnLeuMetLeuMetTrpHisProArgGlnArg                              275280285                                                                     GlyThrAspProThrTyrGlyProAsnGlyCysPheLysAlaLeuAsp                              290295300                                                                     AspIleLeuAsnLeuLysLeuValHisIleLeuAsnMetValThrGly                              305310315320                                                                  ThrIleHisThrTyrProValThrGluAspGluSerLeuGlnSerLeu                              325330335                                                                     LysAlaArgIleGlnGlnAspThrGlyIleProGluGluAspGlnGlu                              340345350                                                                     LeuLeuGlnGluAlaGlyLeuAlaLeuIleProAspLysProAlaThr                              355360365                                                                     GlnCysIleSerAspGlyLysLeuAsnGluGlyHisThrLeuAspMet                              370375380                                                                     AspLeuValPheLeuPheAspAsnSerLysIleThrTyrGluThrGln                              385390395400                                                                  IleSerProArgProGlnProGluSerValSerCysIleLeuGlnGlu                              405410415                                                                     ProLysArgAsnLeuAlaPhePheGlnLeuArgLysValTrpGlyGln                              420425430                                                                     ValTrpHisSerIleGlnThrLeuLysGluAspCysAsnArgLeuGln                              435440445                                                                     GlnGlyGlnArgAlaAlaMetMetAsnLeuLeuArgAsnAsnSerCys                              450455460                                                                     LeuSerLysMetLysAsnSerMetAlaSerMetSerGlnGlnLeuLys                              465470475480                                                                  AlaLysLeuAspPhePheLysThrSerIleGlnIleAspLeuGluLys                              485490495                                                                     TyrSerGluGlnThrGluPheGlyIleThrSerAspLysLeuLeuLeu                              500505510                                                                     AlaTrpArgGluMetGluGlnAlaValGluLeuCysGlyArgGluAsn                              515520525                                                                     GluValLysLeuLeuValGluArgMetMetAlaLeuGlnThrAspIle                              530535540                                                                     ValAspLeuGlnArgSerProMetGlyArgLysGlnGlyGlyThrLeu                              545550555560                                                                  AspAspLeuGluGluGlnAlaArgGluLeuTyrArgArgLeuArgGlu                              565570575                                                                     LysProArgAspGlnArgThrGluGlyAspSerGlnGluMetValArg                              580585590                                                                     LeuLeuLeuGlnAlaIleGlnSerPheGluLysLysValArgValIle                              595600605                                                                     TyrThrGlnLeuSerLysThrValValCysLysGlnLysAlaLeuGlu                              610615620                                                                     LeuLeuProLysValGluGluValValSerLeuMetAsnGluAspGlu                              625630635640                                                                  LysThrValValArgLeuGlnGluLysArgGlnLysGluLeuTrpAsn                              645650655                                                                     LeuLeuLysIleAlaCysSerLysValArgGlyProValSerGlySer                              660665670                                                                     ProAspSerMetAsnAlaSerArgLeuSerGlnProGlyGlnLeuMet                              675680685                                                                     SerGlnProSerThrAlaSerAsnSerLeuProGluProAlaLysLys                              690695700                                                                     SerGluGluLeuValAlaGluAlaHisAsnLeuCysThrLeuLeuGlu                              705710715720                                                                  AsnAlaIleGlnAspThrValArgGluGlnAspGlnSerPheThrAla                              725730735                                                                     LeuAspTrpSerTrpLeuGlnThrGluGluGluGluHisSerCysLeu                              740745750                                                                     GluGlnAlaSer                                                                  755                                                                           __________________________________________________________________________

What is claimed is:
 1. A probe, vector or recombinant nucleic acidcomprising the sequence set forth as SEQ ID NO:
 1. 2. An isolated cellcomprising the probe, vector or recombinant nucleic acid of claim
 1. 3.A probe, vertor or recombinant nucleic acid comprising at least 24consecutive nucleotides of SEQ ID NO: 1, which consecutive nucleotidescomprise nucleotides 72-75 of the sequence set forth as SEQ ID NO:1. 4.An isolated cell comprising the probe, vector or recombinant nucleicacid of claim
 3. 5. The probe, vector or recombinant nucleic acid ofclaim 3 comprising at least 96 consecutive nucleotides of SEQ ID NO:1,which consecutive nucleotides comprise nucleotides 72-75 of the sequenceset forth as SEQ ID NO:1.
 6. An isolated cell comprising the probe,vector or recombinant nucleic acid of claim
 5. 7. A probe, vector orrecombinant nucleic acid encoding a polypeptide comprising the amninoacid sequence set forth as SEQ ID NO:2.
 8. An isolated cell conprisingthe probe, vector or recombinant nucleic acid of claim
 7. 9. A method ofmaking an isolated polypeptide comprising the amino acid sequence setforth as SEQ ID NO:2, said method comprising the steps of:introducingthe vector or recombinant nucleic acid of claim 7 into a host cell orcellular extract, incubating said host cell or cellular extract underconditions whereby said polypeptide is expressed; and isolating saidpolypeptide.
 10. A probe, vector or recombinant nucleic acid encoding apolypeptide comprising at least 10 consecutive amino acid residues ofthe amino acid sequence set forth as SEQ ID NO:2, which consecutiveamino acid residues comprise the amino acid residue 25 of SEQ ID NO:2.11. An isolated cell comprising the probe, vector or recombinant nucleicacid of claim
 10. 12. A method of making an isolated polypeptidecomprising at least 10 consecutive amino acid residues of the amino acidsequence set forth as SEQ ID NO:2, which consecutive amino acid residuescomprise the amino acid residue 25 of SEQ ID NO:2, said methodcomprising the steps of:introducing the vector or recombinant nucleicacid of claim 10 into a host cell or cellular extract, incubating saidhost cell or cellular extract under conditions whereby said polypeptideis expressed; and isolating said polypeptide.
 13. The probe, vector orrecombinant nucleic acid according to claim 10, wherein said polypeptidehas one or more activities selected from the group consisting of: kinaseactivity, kinase inhibitory activity, IκB kinase-α binding activity, IκBkinase-α binding inhibitory activity, IκB kinase-B binding activity, IκBkinase-β binding inhibitory activity, tumor necrosis factorreceptor-associated factor 2 binding activity, tumor necrosis factorreceptor-associated factor 2 binding inhibitory activity, IκB bindingactivity, IκB binding inhibitory activity, nuclear factor-κB activatingactivity and nuclear factor-κB inhibitory activity.
 14. An isolated cellcomprising the probe, vector or recombinant nucleic acid of claim 13.15. A method of making an isolated polypeptide comprising at least 10consecutive amino acid residues of the amino acid sequence set forth asSEQ ID NO:2, which consecutive amino acid residues comprise the aminoacid residue 25 of SEQ ID NO:2, said method comprising the stepsof:introducing the vector or recombinant nucleic acid of claim 13 into ahost cell or cellular extract, incubating said host cell or cellularextract under conditions whereby said polypeptide is expressed; andisolating said polypeptide.